Biochemical assays are routinely used to quantify target molecules in tissue specimen homogenates. Such assays are based on variations in the numbers of normal cells and abnormal cells which express target molecules. IHC assays, in contrast, entail a direct measurement made only on abnormal target molecule expressing cells.
IHC is ideally suited for large sale retrospective studies based on archival material. It provides information not easily obtained by extractive methods of analysis such as the subcellular location of an antigen, heterogeneity of its distribution and correlation with important morphologic parameters. Heretofore, several obstacles have impeded full realization of the advantages theoretically offered by IHC.
IHC quantitation based on paraffin embedded material is difficult, if not impossible. For several reasons, IHC methodology based on visual estimation of the intensity of the immunostaining reaction does not provide a precise, reproducible quantitative assay. Among others, (i) the various tissue processing steps (fixation time, type of fixative, dehydration, embedding and related procedures) each cause some variation, in particular loss of antigen, which modifies the immunostain signal; (ii) immunostain signal strength is a function of tissue section thickness and of staining procedure variables, such as antibody concentration, timing, and chromogen type.